Abstract
The Arg-Gly-Asp (RGD) sequence within the third complementarity-determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin alphaIIbbeta3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (VH domain + Cgamma1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 kappa chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified alphaIIbbeta3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1. 1 Fab molecules bound per platelet was 17% in the presence of 1 m Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 microM Mn2+. The ratio of Fab molecules bound after versus before activation (mean =/- S.D.; n = 3) was: for AP7.3, 9.8 =/- 0.6; for PAC1.1, 8.8 +/- 0.3; and for AP7, 1.4 =/- 0.2. In addition, AP7 bound to the stably expressed integrin mutant alphaIIbbeta3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin alphaIIbbeta3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of alphaIIbbeta3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.
Highlights
¶ To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Inst., 10550 North Torrey Pines Rd., Maildrop SBR13, La Jolla, CA 92037
Ligand size is not a critical factor, because small RGD peptides and peptidomimetics, 5–10-kDa RGD-containing disintegrins, the 150-kDa monoclonal antibody OPG2, and the large 600-kDa tetramer of the murine monoclonal antibody 7E3 all bind to ␣IIb3 whether or not platelet activation has occurred, platelet activation does result in a severalfold increase in apparent binding affinity for some of these ligands [2,3,4,5]
Because the heavy chain CDR3 (H3)1 loops of either AP7 or PAC1.1 contain an RGD motif but differ otherwise in length and flanking amino acid composition, we asked whether differences in amino acid sequence of these flanking regions could be a major factor contributing to the ability of these Fab molecules to distinguish activated from resting conformations of the integrin
Summary
¶ To whom correspondence should be addressed: Dept. of Molecular and Experimental Medicine, The Scripps Research Inst., 10550 North Torrey Pines Rd., Maildrop SBR13, La Jolla, CA 92037. Ligand valency is not a likely explanation for selective binding to activated conformations of ␣IIb3, because the binding of the monoclonal antibody PAC1 to platelets remains dependent upon the activated state of ␣IIb3 whether the antibody is presented as a 50-kDa recombinant Fab molecule or a 900-kDa pentameric IgM [6]. Recombinant PAC1.1 Fab molecules bind more avidly to the activated conformation of ␣IIb3, as reflected by an increase in affinity and number of sites In this regard, the behavior of PAC1.1 is more similar to that of other macromolecular RGD ligands specific for ␣IIb3, e.g. fibrinogen or von Willebrand factor [1, 8, 9].
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